An established hallmark of tumorigenesis is the biosynthesis of aberrant glycan chains due to changes in the expression of glycoprocessing enzymes in tumor tissue. These aberrations become more marked as the tumor acquires a more aggressive phenotype. Tumor cell-surface carbohydrates play important roles in the motility and metastasis of many different cancer cells. In addition, many of these aberrant glycans are tumor-associated carbohydrate antigens (TACA) and have been used in the development of tumor vaccines. Since most of the cellular interactions with TACAs are not well understood, there is an urgent need to better characterize the specific molecular interactions that occur during these events. One feature of carbohydrate binding to macromolecules that is well understood is the concept of multivalency: Monomer carbohydrates bind to proteins very weakly while clustering of a monomer raises this affinity as much as a million-fold. We have prepared the important Thomsen-Friedenreich (Tf) antigen (Gal(beta)1-3GalNAc(alpha)-O-Ser/Thr) on very specific templates to take advantage of this so-called cluster glycoside effect. As mentioned in the last report, we have prepared gold self-assembled nanospheres and quantum dots containing sugar derivative and reported preliminary details on their function. The in vivo experiments with our gold nanospheres in mice were conflicting, so we retreated to basics and performed more rigorous characterization and explored a host of new syntheses that allowed for production of more uniform particles. We proceeded to systematically study the optimum procedure, from several related methods, that offered the highest quality particles with regards to stability and uniformity. We are still examining these data in various media to test for stability. We have prepared the TF antigen in different contexts (attached to both serine and threonine) and linked them to particles. Our TF particles have now been shown in pull down experiments to bind to Galectin-3 and integrin complexes related to metastasis. This work is continuing with our collaborator Vladislav Glinskii, who is interested in defining the TF-glycome, i.e. identify all the proteins in prostate cancer cells that have O-linked TF antigen. We are working on a proteomic method to help him define these molecules in the next year. We put a heavy emphasis on preparing particles that encompassed what we consider the best antigen, a glycopeptide from tumor associated cell-surface mucins, and combined that with various concentrations of linker and T-helper epitope to construct particles that may act as novel immunogens. We prepared at least seven separate particles with various placements of the disaccharide on the peptide, and along with linker and a 28-residue portion of C3d, a domain of complement component 3 and a ligand of CD21, a B-cell surface protein that, when engaged, lowers the threshold of B-cell activation.. These particles were injected into mice and the sera were analyzed for immune responses. A statistically significant immune response was observed in at least two test groups, and animals we boosted a second time with fresh particles. Tumors were implanted and survival was followed. Although one specific antigen group did better than the others, they did not do better than the group that received only PBS. There are several parameters that could have led to a lower than desired reponse, and we are looking into these now. A new study started in collaboration with Howard Young, has us exploring the modulation in cytokine profiles that is elicited by particles with varying antigens in different chemical guises. Initial data is very encouraging as the levels of several cytokines from activated murine macrophages are either potentiated or attenuated with particles containing different surface chemistries. The plan is to reproduce these data, extend this to dendritic cells and explore models for reinjecting these cells back into animals to examine the response to tumor. In addition, TEM experiments are defining the uptake of these particles in mouse macrophages and dendritic cells We also have explored other methods for synthesizing TF and other glycopeptide antigens with optimized linkers to attach to nano-platforms. Novel linker chemistry was developed and the use of lipoic acid-based terminal disulfides as conjugating moieties to gold and other surfaces. Various constructs were prepared to compare the uniformity of the particles with changes in surface chemistry. Finally, we have developed an assay to fully characterize the molecular composition of the particles we prepared. Details of how this can be accomplished in the literature were scant and unsatisfying and we decided to develop our own method. We have worked out procedures for taking small amounts of the nanoparticles, stripping them completely of their ligands and conjugating each to a fluorescent tag that can quantitated by HPLC and fluorescence spectrophotometry. This method has been refined and a manuscript detailing these experiments is being prepared.